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WO2016083458A1 - Modèles de troubles neurologiques du développement basés sur la reprogrammation et utilisations associées - Google Patents

Modèles de troubles neurologiques du développement basés sur la reprogrammation et utilisations associées Download PDF

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Publication number
WO2016083458A1
WO2016083458A1 PCT/EP2015/077659 EP2015077659W WO2016083458A1 WO 2016083458 A1 WO2016083458 A1 WO 2016083458A1 EP 2015077659 W EP2015077659 W EP 2015077659W WO 2016083458 A1 WO2016083458 A1 WO 2016083458A1
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WIPO (PCT)
Prior art keywords
ipsc
syndrome
gtf2i
disorder
genes
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PCT/EP2015/077659
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English (en)
Inventor
Giuseppe Testa
Sina ATASHPAZGARGARI
Antonio ADAMO
Pierre-Luc GERMAIN
Giuseppe MERLA
Giuseppe D'AGOSTINO
Matteo ZANELLA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IRCCS Casa Sollievo Della Sofferenza
Universita degli Studi di Milano
IEO ISTITUTO EUROPEO DI ONCOLOGIA Srl
Istituto Europeo di Oncologia SRL IEO
Original Assignee
IRCCS Casa Sollievo Della Sofferenza
Universita degli Studi di Milano
IEO ISTITUTO EUROPEO DI ONCOLOGIA Srl
Istituto Europeo di Oncologia SRL IEO
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Application filed by IRCCS Casa Sollievo Della Sofferenza, Universita degli Studi di Milano, IEO ISTITUTO EUROPEO DI ONCOLOGIA Srl, Istituto Europeo di Oncologia SRL IEO filed Critical IRCCS Casa Sollievo Della Sofferenza
Priority to ES15801762T priority Critical patent/ES2973286T3/es
Priority to US15/529,886 priority patent/US11369577B2/en
Priority to EP15801762.4A priority patent/EP3224352B1/fr
Publication of WO2016083458A1 publication Critical patent/WO2016083458A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Definitions

  • the present invention relates to iPSC produced from fibroblast obtained from a subject affected by a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ) and uses thereof.
  • the present invention also relates to a cortical neural progenitor cell or a terminally differentiated cortical glutamatergic or gabaergic neuronal cell or a neural crest stem cell line, a mesenchymal stem cell line produced from the iPSC or iPSC line.
  • the invention also relates to method for identifying a compound for the treatment and/or prevention of a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ) and to a LSD1 inhibitor or a HDAC2 inhibitor for use in the treatment of such disorders.
  • ID intellectual disability
  • ASD Autism Spectrum Disorder
  • SZ Schizophrenia
  • iPSC induced pluripotent stem cells
  • iPSC-derived neurons as a higher-throughput readout for autism: promises and pitfalls
  • 2 Prilutsky et al. present an overview of efforts to study iPSC-derived neurons as a model for autism, and explore the plausibility of gene expression profiling as a reproducible and stable disease marker.
  • the elucidation of disease etiologies and establishment of robust, scalable, high-throughput screening assays for autism spectrum disorders (ASDs) have been impeded by both inaccessibility of disease-relevant neuronal tissue and the genetic heterogeneity of the disorder.
  • Neuronal cells derived from induced pluripotent stem cells (iPSCs) from autism patients may circumvent these obstacles and serve as relevant cell models.
  • derived cells are characterized and screened by assessing their neuronal phenotypes. These characterizations are often etiology-specific or lack reproducibility and stability.
  • Ghosh et al. 3 highlight that the rising rates of autism spectrum disorder (ASD) and the lack of effective medications to treat its core symptoms have led to an increased sense of urgency to identify therapies for this group of neurodevelopmental conditions.
  • ASD autism spectrum disorder
  • Developing drugs for ASD has been challenging because of a limited understanding of its pathophysiology, difficulties in modelling the disease in vitro and in vivo, the heterogeneity of symptoms, and the dearth of prior experience in clinical development. In the past few years these challenges have been mitigated by considerable advances in the understanding of forms of ASD caused by single-gene alterations, such as fragile X syndrome and tuberous sclerosis. In these cases they have gained insights into the pathophysiological mechanisms underlying these conditions.
  • iPSCs for ASD drug discovery: patient-derived fibroblasts can be reprogrammed to generate induced pluripotent stem cells (iPSCs) that can be differentiated into neurons to assess cellular phenotypes associated with autism spectrum disorder (ASD).
  • iPSCs induced pluripotent stem cells
  • ASD autism spectrum disorder
  • iPSCs derived from patients with neurodevelopmental disorders exhibit phenotypes distinct from those of cells from healthy volunteers.
  • Muotri and colleagues reported that iPSC-derived neurons from patients with Rett syndrome have a defect in synapse density and network activity 4 .
  • iPSC-derived neurons from patients with Timothy syndrome display electrophysiological defects 5 .
  • WO2011079307 relates to Rett Syndrome as progressive neurological disorder caused by mutations in X- linked gene encoding MeCP2.
  • X-chromosome inactivated female human neural cell derived from an induced pluripotent stem cell are used.
  • a method of identifying a compound useful in treating the neurological disorder is described.
  • WO2013163455 relates to a method for screening candidate drugs that inhibit a neurological disease associated with a MeCP2 mutation, haploid insufficiency or a X-linked gene mutation or aberrant activity comprising inducing iPSC from a male subject to undergo neuronal differentiation and analyzing treated cells for an increase in neuronal networks, dendritic spine density, synapses, soma size, neuronal excitation, or calcium signaling.
  • US2007218068 relates to the identification of a human autism susceptibility gene, which can be used for the diagnosis, prevention and treatment of autism and related disorders, as well as for the screening of therapeutically active drugs. The document describes that the ⁇ 1 gene on chromosome 5 and certain alleles thereof are related to susceptibility to autism and represent novel targets for therapeutic intervention.
  • CNV symmetrical copy number variations
  • alterations are then selectively amplified upon differentiation into disease-relevant lineages, thereby establishing the value of large iPSC cohorts in the elucidation of disease-relevant developmental pathways.
  • the inventors functionally define the quota of transcriptional dysregulation specifically caused by dosage imbalances in GTF2I, a transcription factor in 7qll.23 thought to play a critical role in the two conditions, which the inventorsfound associated to key repressive chromatin modifiers.
  • WBS Williams-Beuren syndrome
  • OMIM 194050 Williams-Beuren region duplication syndrome
  • 7dupASD autistic spectrum disorder 8 .
  • WBS and 7dupASD involve, respectively, the loss or gain of 26-28 genes and have a prevalence of between 1 in 7,500 and 1 in ⁇ , ⁇ 9,10 .
  • WBS is characterized by cardiovascular symptoms and facial dysmorphism, along with the hallmark behavioral-cognitive profile that combines hypersociability with comparatively well-preserved language abilities, but severely compromised visuo-spatial processing, counting and planning 9 ' 11 .
  • 7dupASD in contrast, features varying degrees of ASD ranging from severe speech impairment to full blown autism, along with craniofacial dysmorphisms, among which some are similar and some symmetrically opposite to those of WBS patients 10 ' 12 .
  • both syndromes are associated with anxiety and attention deficit hyperactivity disorder (ADHD).
  • ADHD attention deficit hyperactivity disorder
  • the two conditions are paradigmatic of a fundamental aspect of CNV-based disease pairs, namely the fact that symmetrically opposite CNV result in shared as well as symmetrical phenotypes. Yet, despite significant insight from mouse models 13"16 , the molecular pathways specifically affected by 7ql 1.23 CNV in the human lineages that are most relevant for disease phenotypes are yet to be uncovered.
  • the inventors present the largest cohort of WBS and 7dupASD iPSC lines and differentiated lineages, in which the inventors find that 7qll.23 dosage impacts disease- relevant transcriptional programs already in the pluripotent state. These alterations are partitioned into shared and symmetrically opposite ones and are further exacerbated upon differentiation into disease-relevant lineages. Finally, the inventors dissect the specific contribution of transcriptional factor GTF2I (also known as TFII-I), a key 7ql 1.23 gene, to the molecular phenotype of the two conditions in early developmental lineages.
  • GTF2I also known as TFII-I
  • the present invention provides an induced pluripotent stem cell (iPSC) or an iPSC line produced from a fibroblast obtained from a subject wherein said iPSC or an iPSC line expresses detectable levels of at least one of the following markers: OCT4, SOX2, NANOG, KLF4 and wherein the subject is affected by a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
  • ID neurodevelopmental disorder entailing intellectual disability
  • ASD Autism Spectrum Disorder
  • SZ Schizophrenia
  • the subject is affected by either one of the two disorders caused by symmetrical copy number variations (CNV) at 7qll.23, namely Williams Beuren syndrome (WBS, OMUM
  • 7qll.23 microduplication syndrome also known as Somerville-van der Aa syndrome, OMIM 609757
  • autistic spectrum disorder hereafter 7dupASD
  • iPSC or iPSC lines are generated using modified mRNA which enables non-integrating reprogramming of cells.
  • the cells of the invention are mammal cells, preferably human cells.
  • said iPSC or iPSC line harbors at least one genetic change as described in Table 4.
  • Such change may be introduced during the reprogramming process and renders these cells genetically distinct from any other somatic cell naturally existing in the subject from which they derive.
  • the iPSC or iPSC line may be engineered and genetically modified. Any method known in the art used to engineer cells may be used.
  • said iPSC or iPSC line expresses a reporter gene under the control of a dorsal telencephalon stem cell gene promoter.
  • the dorsal telencephalon stem cell gene promoter is selected from the group consisting of the promoters of: FOXG1, PAX6, EGR1, FOS and TBR2.
  • said iPSC or iPSC line stably expresses NGN2.
  • Preferably said stable expression of NGN2 is inducible.
  • the stable expression of NGN2 may be achieved by any means known in the art such as genetic modification, transfection, exogenous exposure ect.
  • the present invention provides the iPSC or iPSC line as defined abocve for use in a method to purify and/or isolate a cortical neural progenitor cell or a neuronal cell.
  • the method to purify and/or isolate a cortical neural progenitor cell or a neuronal cell comprises: i) inducing neurulation of a subject iPSC and generate neural rosettes following established protocols; ii) purifying cortical progenitor cells expressing at least one of the markers: FOXG1, PAX6, EGR1, FOS, TBR2 by either durg selection or fluorescence activated cell sorting (FACS) enabled by the respective reporter constructs; iii) further differentiating said purified neural progenitors into postmitotic, terminally differentiated neuron.
  • FACS fluorescence activated cell sorting
  • the present invention provides a population comprising two or more iPSC or iPSC line of the invention.
  • the present invention provides cortical neural progenitor cell produced from the iPSC or iPSC line of the invention, wherein said cortical neural progenitor cell expresses detectable levels of at least one of the following markers: FOXG1, PAX6, EGR1, FOS and TBR2, ZOl, SOX2.
  • the present invention provides a terminally differentiated cortical glutamatergic neuronal cell produced from the iPSC or iPSC line according to the invention, directly or through the intermediate of the cortical neural progenitor cell of the invention, wherein said terminally differentiated cortical glutamatergic neuronal cell expresses detectable levels of at least one of the following markers: PSD95, Synapsin, Vglut, Ctip2, BRN2, TBR1, TBR2, Cux 1, Reelin, Satb2, MAP2, TUJ1 Classin, Glu-Rl, ER81, vGlutl, Homerl, Homer 2, Synaptophysin, Foxpl, GRINl, GRIN2D, GRIN2A, GRM1, GRM8, slcla6 , slcl7a7, TrkB.
  • markers PSD95, Synapsin, Vglut, Ctip2, BRN2, TBR1, TBR2, Cux 1, Reelin, Satb2, MAP2,
  • the present invention provides a terminally differentiated cortical gabaergic neuronal cell produced from the iPSC or iPSC line according to the invention, directly or through the intermediate of the cortical neural progenitor cell of the invention, wherein said terminally differentiated cortical gabaergic neuronal cell expresses detectable levels of at least one of the following markers: Nkx2.1, GABA, VGAT, ASCL1, DLX2, DLX5, CALBINDUN, CALRETHNIN, SOMATOSTATIN, PARVALBUMIN, GAD67, SLC32A1, PSD95, Synapsin, Vglut, Ctip2, BRN2, TBR1, TBR2, Cux 1, Satb2, MAP2, TUJ1 Classin, Glu-Rl, vGlutl, Homerl, Synaptophysin.
  • the present invention provides a population comprising two or more cortical neural progenitor cells according to the invention.
  • the present invention provides a population comprising two or more terminally differentiated cortical glutamatergic or gabaergic neuronal cells according to the invention.
  • the present invention provides neural crest stem cell line produced from the iPSC or iPSC line according to any one of claim 1 to 7, wherein said neural crest stem cell expresses detectable levels of at least one of the following markers: HNK1, NGFR, SOX9, SOX10, PAX3, ZINC1 and TFAP2.
  • the present invention provides a population comprising two or more neural crest stem cells as defined above.
  • the present invention provides a mesenchymal stem cell line produced from the iPSC or iPSC line as defined above, wherein said mesenchymal stem cell expresses detectable levels of at least one of the following markers: CD44 and CD73.
  • the present invention provides a population comprising two or more mesenchymal stem cells as defined above.
  • the present invention provides a method to obtain iPSC or iPSC line as defined above and stably expressing NGN2 comprising the steps of:
  • the method comprises the steps of:
  • iPSCs as DAPI-negative single cells, preferably in 96 well plates;
  • the present invention provides a method for identifying a compound for the treatment and/or prevention of a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ) comprising:
  • the present screening platform/method is primarily designed to screen for gene dosage rescue at the mRNA level, measured for instance by qRT-PCR.
  • the present invention's screening is primarily designed at finding compounds that rescue the dosage of critical genes involved in a neurodevelopmental disorder entailing intellectual disability (ID) and/or a disorder belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
  • ID neurodevelopmental disorder entailing intellectual disability
  • ASD Autism Spectrum Disorder
  • SZ Schizophrenia
  • Measurement of expression can be performed by multiplex quantitative Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) or barcode-enabled high-throughput Ribonucleic Acid (RNA) sequencing;
  • RT quantitative Reverse Transcription
  • PCR Polymerase Chain Reaction
  • RNA Ribonucleic Acid
  • Preferably said method involves HTS-format differentiation starting from previously generated cortical neural progenitors as described above.
  • the method further comprises analyzing iPSC-derived neurons for morphology (dendritic arborization, density and shape of dendritic spines), expression of markers of neuronal differentiation, synaptic maturation and function.
  • morphology dendritic arborization, density and shape of dendritic spines
  • the analyzis of iPSC-derived neurons may be cariied out either directly or thorugh the intermediate of cortical neural progenitors and may including HTS-compatible quantitation of dendritic arborization, density and shape of dendritic spines, expression of markers of neuronal differentiation, synaptic maturation and function (including eitehr post-fixation assessment of defining marekrs or live- imaging of enuronal activity-dependent calcium signaling).
  • the Autism Spectrum Disorder is WBS or 7dupASD. Thanks to the finding of the present invention, it is also provided a LSD1 inhibitor or a HDAC2 inhibitor for use in the prevention and/or treatment of a neurodevelopmental disorder entailing intellectual disability (ID) and/or an Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
  • a neurodevelopmental disorder entailing intellectual disability (ID) and/or an Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
  • the Autism Spectrum Disorder (ASD) is WBS or 7dupASD and the Schizophrenia (SZ) is a 7ql 1.23 microduplication-dependent Schizophrenia (SZ).
  • an autistic pathology means a condition matching at least one criterion in the definition of the Autism Spectrum Disorder (ASD), including the conditions associated to one of the following genetic lesions.
  • ASD and SZ pathologies are comprised in the following:
  • srxy9 46,xy sex reversal 9; srxy9 (616067). abelson helper integration site 1; ahil (608894). acrodysostosis 2 with or without hormone resistance; acrdys2 (614613). activity-dependent neuroprotector homeobox; adnp (611386). adenosine deaminase; ada (608958). adenylosuccinase deficiency (103050). aldehyde dehydrogenase 1 family, member a3; aldhla3 (600463). aldehyde dehydrogenase 5 family, member al; aldh5al (610045). algl2, s.
  • asperger syndrome susceptibility to, 1; aspgl (608638). asperger syndrome, susceptibility to, 2; aspg2 (608631). asperger syndrome, susceptibility to, 3; aspg3 (608781). asperger syndrome, susceptibility to, 4; aspg4 (609954).
  • asperger syndrome x-linked, susceptibility to, 1; aspgxl (300494).
  • asperger syndrome x- linked, susceptibility to, 2; aspgx2 (300497). athabaskan brainstem dysgenesis syndrome; abds bosley-salih-alorainy syndrome, included; bsas, included (601536).
  • autism autism susceptibility to, 1, included; autsl, included (209850). autism, susceptibility to, 10; autslO (611016). autism, susceptibility to, 11; autsl 1 (610836). autism, susceptibility to, 12; autsl 2 (610838). autism, susceptibility to, 13; autsl3 (610908).
  • autism susceptibility to, 15; autsl5 (612100). autism, susceptibility to, 16; autsl6 (613410). autism, susceptibility to, 17; autsl7 (613436). autism, susceptibility to, 18; autsl8 (615032). autism, susceptibility to, 19; autsl9 (615091). autism, susceptibility to, 3; auts3 (608049). autism, susceptibility to, 5; auts5 (606053). autism, susceptibility to, 6; auts6 (609378). autism, susceptibility to, 7; auts7 (610676). autism, susceptibility to, 8; auts8 (607373).
  • autism susceptibility to, 9; auts9 (611015). autism, susceptibility to, x-linked 1; autsxl (300425). autism, susceptibility to, x-linked 2; autsx2 mental retardation, x-linked, included (300495). autism, susceptibility to, x-linked 3; autsx3 (300496). autism, susceptibility to, x-linked 5; autsx5 (300847). bannayan-riley-ruvalcaba syndrome; brrs (153480). beckwith-wiedemann syndrome; bws beckwith-wiedemann syndrome chromosome region, included; bwcr, included (130650).
  • brachydactyly-mental retardation syndrome bdmr chromosome 2q37 deletion syndrome, included (600430). branched-chain alpha-keto acid dehydrogenase kinase; bckdk (614901). branched-chain ketoacid dehydrogenase kinase deficiency; bckdkd (614923). brunner syndrome (300615). cadherin 8; cdh8 (603008). calcium channel, voltage-dependent, 1 type, alpha- lc subunit; cacnalc (114205). calcium channel, voltage-dependent, 1 type, alpha-Id subunit; cacnald (114206).
  • chromosome 15qll-ql3 duplication syndrome autism susceptibility to, 4, included; auts4, included (608636).
  • chromosome 15qll.2 deletion syndrome (615656).
  • chromosome 15ql3.3 deletion syndrome (612001).
  • chromosome 15q25 deletion syndrome (614294).
  • chromosome 16pl 1.2 deletion syndrome 220-kb body mass index quantitative trait locus 16, included; bmiql6, included (613444).
  • chromosome 16pll.2 deletion syndrome 593-kb autism, susceptibility to, 14a, included; autsl4a, included (611913).
  • chromosome 16pl 1.2 duplication syndrome autism susceptibiUty to, 14b, included; autsl4b, included (614671).
  • chromosome 16pl2.1 deletion syndrome 520-kb fragile site 16pl2, included (136570).
  • chromosome 16pl2.2-pll.2 deletion syndrome 7.1- to 8.7-mb (613604).
  • chromosome 16pl3.3 deletion syndrome proximal (610543).
  • chromosome 16pl3.3 duplication syndrome (613458).
  • chromosome 17ql2 deletion syndrome (614527).
  • chromosome 17q21.31 duplication syndrome (613533).
  • chromosome 1 q21.1 deletion syndrome 1.35-mb (612474).
  • chromosome 1 q21.1 duplication syndrome (612475).
  • chromosome 22qll.2 duplication syndrome (608363).
  • chromosome 2pl6.1-pl5 deletion syndrome (612513).
  • chromosome 2pl6.3 deletion syndrome schizophrenia 17, included; sczdl7, included (614332).
  • chromosome 3pter-p25 deletion syndrome (613792).
  • chromosome 3ql3.31 deletion syndrome (615433).
  • chromosome 3q29 deletion syndrome (609425).
  • chromosome 4q32.1- q32.2 triplication syndrome (613603).
  • chromosome xp22 deletion syndrome (300830). cognitive function 1, social; cgfl (300082). contactin 4; cntn4 (607280). contactin-associated protein-like 2; cntnap2 (604569). Cornelia de lange syndrome 1; cdlsl (122470). cortactin- binding protein 2; cttnbp2 (609772). cowden syndrome 1; cwsl dysplastic gangliocytoma of the cerebellum, included (158350). craniosynostosis 3; crs3 (615314). cyclic vomiting syndrome; cvs cyclic vomiting syndrome with neuromuscular disease, included (500007).
  • cyclin-dependent kinase inhibitor lb cyclin- dependent kinase inhibitor lb; cdknlb (600778). cystinuria cystinuria, type a, included (220100).
  • dcnl domam-containing protein 1 dcunldl (605905). diamond-blackfan anemia 6; dba6 (612561).
  • dmydropyrimidine dehydrogenase deficiency 5-@fluorouracil toxicity included (274270).
  • distal-less homeobox 2; dlx2 (126255). doublecortin; dcx (300121).
  • epilepsy familial focal, with variable foci; ffevf (604364). epilepsy, focal, with speech disorder and with or without mental retardation; fesd landau-kleffher syndrome, included; Iks, included (245570). epilepsy, x-linked, with variable learning disabilities and behavior disorders (300491). epileptic encephalopathy, childhood-onset; eeoc (615369). epileptic encephalopathy, early infantile, 1; eieel (308350).
  • epileptic encephalopathy early infantile, 13; eieel3 (614558). epileptic encephalopathy, early infantile, 6; eiee6 (607208). epileptic encephalopathy, early infantile, 9; eiee9 (300088). epsilon-trimethyllysine hydroxylase deficiency; tmlhed (300872). epsilon- trimethyllysine hydroxylase; tmlhe (300777). eukaryotic translation initiation factor 4e-bmding protein 2; eif4ebp2 (602224). eukaryotic translation initiation factor 4e; eif4e (133440).
  • frnrl gene frnrl fragile site, folic acid type, rare, fraxq27.3, included; fraxa, included (309550). forkhead box pi; foxpl (605515). forkhead box p2; foxp2 (605317). fragile x mental retardation syndrome primary ovarian insufficiency, fragile x-associated, included (300624). fragile x tremor/ataxia syndrome; fxtas (300623). gamma-aminobutyric acid receptor, alpha-4; gabra4 (137141). gamma-aminobutyric acid receptor, alpha-5; gabra5 (137142).
  • gamma- aminobutyric acid receptor beta-3; gabrb3 (137192).
  • gamma-aminobutyric acid receptor gamma-3; gabrg3 (600233). gastrin-releasing peptide receptor; grpr (305670).
  • gephyrin gphn mll/gphn fusion gene, included (603930).
  • gilles de la tourette syndrome gts chronic motor tics, included (137580).
  • glutamate receptor ionotropic, n-methyl-d-aspartate, subunit 2b; grin2b (138252).
  • glutamate receptor metabotropic, 5; grmS (604102).
  • glycine encephalopathy gee hyperglycinemia, transient neonatal, included; tnh, included (605899).
  • glyoxalase i glol (138750).
  • hect domain and reel -like domain 2 herc2 (605837).
  • hepatocyte cell adhesion molecule hepacam (611642).
  • homeobox al hoxal (142955).
  • homer drosophila, homolog of, 1; homer 1 homer la, included (604798).
  • hyper-ige recurrent infection syndrome autosomal dominant (147060).
  • hyperlexia (238350).
  • interleukin 1 receptor accessory protein-like 1 illrapll illrapll/dmd fusion gene, included (300206).
  • microphthalmia isolated 3; mcop3 (611038). microphthalmia, isolated 8; mcop8 (615113). microphthalmia, syndromic 1; mcopsl (309800).
  • microtubule- associated protein 2 map2 (157130). mlcl gene; mlcl (605908). modifier, x-linked, for neurofunctional defects (309840). mo mo syndrome (157980). monoamine oxidase a; maoa antisocial behavior following childhood maltreatment, susceptibility to, included (309850). mucopolysaccharidosis, type iiib; mps3b (252920). myhre syndrome; myhrs (139210). n- acetylglucosaminidase, alpha-; naglu (609701). nance-horan syndrome; nhs (302350). necdin- like gene 2; ndnl2 (608243).
  • neuroligin 4 y-linked; nlgn4y (400028).
  • phelan-mcdermid syndrome (606232). phosphatase and tensin homolog; pten pten hamartoma tumor syndrome, included; phts, included (601728).
  • potassium channel voltage-gated, subfamily h, member 5; kcnh5 (605716).
  • potassium voltage-gated channel shal-related subfamily, member 2; kcnd2 (605410).
  • potocki- lupski syndrome ptls (610883). prader-willi-like syndrome; pwls (615547).
  • ribosomal protein 110 ribosomal protein 110; rpllO (312173). ricMeri-cosWguion-almeida syndrome (268850).
  • semaphorin 5a; sema5a 609297).
  • solute carrier family 35 (udp-n-acetylglucosamine transporter), member 3; slc35a3 (605632). solute carrier family 6 (neurotransmitter transporter, serotonin), member 4; slc6a4 (182138). solute carrier family 9 (sodium/hydrogen exchanger), member 9; slc9a9 (608396).
  • specific language impairment 3 sli3 (607134).
  • specific language impairment 4; sli4 (612514).
  • specific language impairment 5; sli5 (615432).
  • spchl 602081.
  • succinic semialdehyde dehydrogenase deficiency ssadhd (271980).
  • suppressor of tumorigenicity 7 st7 (600833).
  • synapsin i synl (313440).
  • synaptic ras-gtpase-activating protein 1 syngapl (603384).
  • temtamy syndrome temtys (218340).
  • thioredoxin reductase 2; txnrd2 606448).
  • timothy syndrome ts (601005).
  • topoisomerase dna, i; topi topl/nup98 fusion gene, included (126420). topoisomerase, dna, ii, beta; top2b (126431). tscl gene; tscl (605284). tsc2 gene; tsc2 (191092). tuberous sclerosis 1; tscl (191100). tuberous sclerosis 2; tsc2 tsc2 angiomyolipomas, renal, modifier of, included (613254). tyrosine hydroxylase; th (191290). ubiquitin-protein ligase e3a; ube3a (601623).
  • catechol-o-methyltransferase catechol-o-methyltransferase
  • comt catechol- o-methyltransferase activity low, in red cells, included (116790).
  • reticulon 4 receptor; rtn4r solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter), member 1; slclal (133550).
  • chromosome 1 q21.1 duplication syndrome (612475). chitinase 3-like 1; chi311 (601525). apolipoprotein 1-iv; apol4 (607254). chromosome 1 q21.1 deletion syndrome, 1.35-mb (612474). d-amino acid oxidase; dao (124050). neuregulin 1; nrgl glial growth factor 2, included; ggf2, included (142445). apolipoprotein 1-ii; apol2 (607252). chromosome 17ql2 deletion syndrome (614527).
  • darier-white disease dar darier disease, acral hemorrhagic type, included (124200).
  • daoa antisense rna; daoaas 607415).
  • atp-binding cassette subfamily a, member 13; abcal3 (607807). deafness, autosomal recessive 47; dfhb47 (609946).
  • dopamine receptor d4; drd4 (126452).
  • synapsin iii; syn3 602705). protein phosphatase 3, catalytic subunit, gamma isoform; ppp3cc (114107).
  • potassium channel calcium-activated, intermediate/small conductance, subfamily n, member 3; kcnn3 (602983). serine racemase; srr (606477).
  • chromosome 16pl3.3 deletion syndrome, proximal 610543).
  • n-ethylmaleimide-sensitive factor; nsf 601633).
  • clathrin interactor 1 clintl (607265).
  • translin-associated factor x tsnax (602964).
  • glutamate-cysteine ligase modifier subunit; gclm (601176).
  • notch drosophila, homolog of, 4; notch4 mouse mammary tumor virus integration site 3, included (164951).
  • v- erb-b2 avian erythroblastic leukemia viral oncogene homolog 4; erbb4 (600543).
  • chromosome 16pl 1.2 duplication syndrome autism susceptibility to, 14b, included; autsl4b, included (614671).
  • chromosome 22qll.2 duplication syndrome 608363).
  • kiaa0391 gene kiaa0391 (609947).
  • g protein-coupled receptor 50; gpr50 (300207).
  • oligodendrocyte lineage transcription factor 2 oligodendrocyte lineage transcription factor 2; olig2 (606386). velocardiofacial syndrome (192430). major affective disorder 1; mafdl (125480). glutamate-cysteine ligase, catalytic subunit; gclc (606857). reelin; rein (600514). fasciculation and elongation protein zeta 1; fezl (604825). chromosome 16pl 1.2 deletion syndrome, 593-kb autism, susceptibility to, 14a, included; autsl4a, included (611913). digeorge syndrome critical region gene 8; dgcr8 (609030).
  • hsr monoamine oxidase b; maob (309860). quaking, mouse, homolog of; qki (609590). chromosome 3q29 deletion syndrome (609425). ataxin 1; atxnl atxnl, alternative reading frame, included (601556). abelson helper integration site 1; ahil (608894). congenital anomalies of kidney and urinary tract, susceptibility to; cakut (610805). dna methyltransferase 1; dnmtl (126375). rel-silencing transcription factor; rest (600571).
  • v-src avian sarcoma (schmidt-ruppin a-2) viral oncogene; src (190090). autism autism, susceptibility to, 1, included; autsl, included (209850). drug metabolism, poor, cyp2d6-related drug metabolism, ultrarapid, cyp2d6-related, included (608902). metachromatic leukodystrophy pseudoarylsulfatase a deficiency, included (250100). major depressive disorder; mdd seasonal affective disorder, included; sad, included (608516). pericentriolar material 1; pcml pcml/ret fusion gene, included (600299).
  • potassium channel voltage-gated, subfamily h, member 2; kcnh2 (152427). muscular dystrophy, becker type; bmd (300376). apolipoprotein 1-i; apoll (603743). retinoic acid-induced gene 1; rail (607642). solute carrier family 6 (neurotransmitter transporter, serotonin), member 4; slc6a4 (182138). dopamine beta-hydroxylase, plasma; dbh (609312). ciliary neurotrophic factor; cntf (118945). proopiomelanocortin; pome melanotropin, included (176830). amyloid beta a4 precursor protein; app (104760).
  • synaptosomal-associated protein 25-kd; snap25 (600322). hashimoto thyroiditis thyroid autoantibodies, included (140300).
  • frontotemporal dementia; ftd pick complex included (600274). neuropeptide y; npy (162640). t-box 1; tbxl (602054). phelan-mcdermid syndrome (606232). solute carrier family 6 (neurotransmitter transporter, dopamine), member 3; slc6a3 (126455). ataxin 8 opposite strand; atxn8os (603680). circadian locomotor output cycles kaput; clock (601851). ferritin light chain; ftl (134790). orofacial cleft 1; ofcl (119530). wfsl gene; wfsl (606201).
  • arylsulfatase a arsa (607574). nuclear receptor subfamily 0, group b, member 1; nrObl (300473). huntington disease; hd (143100). alzheimer disease; ad alzheimer disease, familial, 1, included; adl, included (104300). apolipoprotein 1-vi; apol6 (607256). apolipoprotein 1-v; apol5 (607255). heparan sulfate 6-o-sulfotransferase 3; hs6st3 (609401). micro rna 130b; mirl30b (613682). trace amine-associated receptor 5; taar5 (607405).
  • rogdi drosophila, homolog of; rogdi (614574). homer, drosophila, homolog of, 2; homei2 homei2a, included (604799).
  • ASD and SZ may be caused by CNV and or point mutations in a large number of genes or loci (currently adding up to the 275 loci or genes listed above), the core symptoms of ASD and SZ are very similar regardless of the causing CNV/mutation. This suggests a high degree of molecular convergence in the pathological mechanism and supports much current effort in defining nodes of deregulation that, while originally identified in a specific ASD or SZ subset, may obtain also in other forms of either ASD or SZ even when caused by a different genetic lesion 17"20 .
  • a cortical neural progenitor cell means a cell derived from human iPSC upon differentiation into the neural lineage, and expressing at least one of the following defining markers: PAX6, SOX2, FOXG1, OTX2, CDC42, RAC1, ZOl.
  • a neural crest stem cell means a cell derived from human iPSC upon differentiation into the neural crest lineage, and expressing at least both of the following defining markers: HNK1 andNGFR.
  • a mesenchymal stem cell means a cell derived from human iPSC upon differentiation into the mesenchymal lineage, and expressing at least both of the following defining markers: CD44 and CD73.
  • the step c) of measuring the level of expression of at least one gene as defined in the claim may be performed by measuring mRNA by qRT-PCR or by high throughout, barcode-enabled RNA sequencing or by any other method known in the art.
  • control level may be the level of expression of said gene in: i) a neuronal cell derived from an iPSC reprogrammed from a healthy individual (ie.not carrying 7qll.23 CNV); ii) an isogenic control neuronal cell; iii) a mock treated 7dupASD or WBS iPSC.
  • analyzing neural differentiated iPSC-derived cells for morphology and/or function means quantitatively assessing neuronal networks, dendritic spine density, synapses, soma size, neuronal excitation, or calcium signaling, electrophysiological parameters.
  • Neurodevelopmental disorders encompass a wide range of human diseases characterized by abnormal development of the nervous system, especially the cortex.
  • Autism Spectrum Disorders (ASD), Schizophrenia (SZ) and intellectual disability (ID) are the main neurodevelopmental disorders, displaying specific as well as shared domains of deficit.
  • a LSD1 inhibitor is any known LSD1 inhibitor, for instance described in WO2013057322, WO2011131576, WO2014086790, WO2012135113 and applications EP 14170656.4, EP14193312.7 and EP 14183755.9, included by reference.
  • LSD1 inhibitor for instance described in WO2013057322, WO2011131576, WO2014086790, WO2012135113 and applications EP 14170656.4, EP14193312.7 and EP 14183755.9, included by reference.
  • the general formula and the various definition of substituent, including preferred embodiments disclosed in such documents are part of the present application.
  • a HDAC2 inhibitor is any known HDAC2 inhibitor, for instance those described in Dokmanovic M et al. 21 and Kim HJ et al. 22 .
  • Figure 1 Patient cohort and expression of 7qll.23 gene interval according to genotype (a)
  • FIG. 3 GTF2I protein complex and its genome-wide occupancy
  • GTF2I distribution across all genes that are bound in all samples of a given genotype (e) Distribution of GTF2I core targets according to their expression level, showing for each expression range the position of the peak relative to Transcription Start Site (TSS).
  • TSS Transcription Start Site
  • Figure 4 GTF2I represses BEND4 in a dosage-dependent manner, (a) BEND4 expression in iPSC measured by RNA-seq. (b) BEND4 expression in all iPSC lines measured by RT-qPCR. Error bars represent variation between lines of each genotype, and stars indicate statistical significance according to a two-tailed T-test (*: p ⁇ 0.05, **: p ⁇ 0.01, ***: p ⁇ 0.001).
  • RT-qPCR validation of GTF2I knockdowns using different short hairpins (sh2, sh52 and sh08) on at least two lines per genotype, (d) RT-qPCR validation of BEND4 mRNA levels in the sh2, sh52, sh08-GTF2I knock-down cell lines, (e) RNA-seq measurement of BEND4 expression in the same cell lines, (f) RT-qPCR measurement of BEND4 upon irreversible LSD1 inhibition starting at day 0 (DO) and day 4 (D4) of embryoid body formation in a control iPSC line, showing that in both stages, both inhibitors lead to the upregulation of BEND4. All RT-qPCR levels are reported as percentages of GAPDH.
  • RA retinoic acid.
  • IEO-DDP IEO Drug Discovery Program In panels c-d-f, error bars represent variation between 2 technical replicates. Similar results were obtained with the Vimentin gene (see Fig. 10).
  • Figure 5 Derivation and transcriptional characterization of disease-relevant lineages
  • iPSC-derived cortical stem/progenitor cells recapitulate the emergence of stem cell populations in human corticogenesis. Rosettes were stained for proliferating (Ki67), mitotic (phospho- histone H3) and neural stem cell (NESTIN, ZOl, and PAX6) markers (above). Default fbrebrain specification is evidenced by the expression of OTX2, FOXG1 and SOX2 markers (below), (c) Top enrichments for GO biological processes among NPC DEGs.
  • Figure 6 Lineage-specific retention of iPSC DEGs.
  • Figure 7 (a) Graphical representation of the lineage specific retention of DEGs.
  • HDF Human Derived Fibroblast
  • FIG. 8 An approach to isolate iPSC-derived FOXGl-expressing cortical progenitors.
  • the figure describes the proof of principle of experiments aimed at isolating cortical progenitors by selection and FACS sorting. Briefly, the inventors designed a lentiviral construct (upper left scheme) that expresses GFP from a ubiquitous promoter and the puromycinr esistance gene under the control of the FOXG1 promoter.
  • the panels on the right show the infection of a control iPSC line (reprogrammed from a WBS patient relative) with different concentrations of this lentiviral vector, and the assessment of infection efficiency by GFP fluorescence (both by immunofluorescence and FACS, respectively upper and lower panels).
  • FIG. 9 qRT-PCR-based scoring assay for HTS screening.
  • the figure shows the automated workflow the inventors already validated, for the scoring of the expression of GTF2I and BAZ1B (by multiplex qRT-PCR) in iPSC grown on a 96-well format.
  • the upper left panel shows that, with a very robust 5 replicates test, the inventors were able to measure consistently the levels of GTF2I across 3 different concentrations of seeded iPSC (from 6.000 to 12.000), finding the expected linear relationship between number of cells and GTF2I expression, with excellent consistency across different wells of the same plate, (b) The upper right panel shows that even across wells from different plates qRT-PCR measurements are extremely consistent. The middle right panel shows that expression of both GTF2I and BAZ1B can be very reproducibly measured across 2 different plates seeded with iPSC reprogrammed from WBS patients.
  • FIG. 10 (a) GTF2I-mediated BEND4 regulation is dependent on LSD1. (upper panel) BEND4 levels are incraesed upon LSD1 inhibition using two LSD1 irreversible inhibitors to a final concentration of 5uM (Oryzon) and lOuM (DDP26095). Treatments were performed for 5 days at day 0 and day 4 of a Embryoid Bodies (EBs) formation assay. Similar results were obtained for ICAM1 and VIM (vimentin) without (Lower panel left) or in the presence of Retinoic Acid (RA), added to a final concentration of lOuM for 5 days (Lower panel right), (b) GTF2I and LSD1 negatively regulate target genes expression.
  • EBs Embryoid Bodies
  • Proposed model representing the mechanism of action of the GTF2I/LSD1 complex on target genes (BEND4 is shown as a representative target).
  • the GTF2I-LSD1 complex represses target genes by binding to their promoter regions, as exemplified in the upper panel when a control scrambled sh RNAi hairpin is administered to the cells.
  • the middle panel shows relief from transcriptional repression upon GTF2I knock-down accomplished with a specific sh RNAi hairpin targeting GTF2I.
  • the lower panel shows the same result obtained by chemical LSD1 inhibition with the two inhibitors shown in Fig. 10a.
  • FIG. 11 Establishment of clonal NGN2-transduced inducible iPSC lines,
  • (a) Schematic representation of the strategy to establish clonal lines in which the NGN2 transgene and its coactivator are stably integrated by lentiviral transduction. Infected iPSC lines are expanded, sorted as single cells to create clones, and subsequently 3-5 clones per line are tested for GFP activation. GFP-positive clones are then expanded and frozen for subsequent large-scale differentiation experiments. Genomic DNA can be extracted to quantify the number of integration events across clones by digital PCR.
  • (b) The GFP signal can be seen from day 2 after induction, whereas at day 21 induced clonal NGN2-transduced lines express mature neuronal markers such as MAP2, TUJ1 and the glutamatergic marker VGLUT.
  • FIG. 12 iPSC lines derivation, (a) Schematic representation of mRNA mediated reprograrnming. (b) GFP tracking of mesenchymal-to-epithelial transition, (c) For each genetic condition, expression of alkaline phosphatase (ALP) by immunohistochemistry, immunofluorescence for pluripotency markers, and staining of three representative iPSC- derived teratomas expressing markers specific for the three germ layers (right). H&E: Hematoxylin&Eosin. DES: desmin. CK: cytokeratin. (d) Nanostring measurements for pluripotency markers. FOV: field of view.
  • ALP alkaline phosphatase
  • FIG. 13 Expression of genes of the WBS region in iPSC.
  • Figure 14 Transcriptional profiling of patient- and control-derived iPSCs.
  • (a-b-c) GO biological processes enriched among DEGs between Control vs 7dupASD (a), WBS vs Control (b) and WBS vs 7dupASD (c).
  • (d) Enrichment for GO biological processes among the union of DEGs when excluding the external control lines from the analysis.
  • Figure 15 Genes differentially expressed in a symmetrical manner in WBS and 7dupASD iPSC.
  • Figure 16 Comparison of different antibodies assayed for ChlP-Seq and Immunoprecipitation assays, (a) Western blot validation of irnmunoprecipitation efficiency of two different GTF2I antibodies in a control iPSC line, (b) Most gene targets identified with the Bethyl antibody are also identified with the other antibodies, (c) Enrichment plot showing the distribution of reads across the genome; the samples using the Bethyl antibodies have a distinctively greater degree of enrichment compared with the other samples, (d) ChlP-qPCR validation of core and dosage-sensitive GTF2I targets.
  • EOMES and SNAP25 promoters have been used as negative controls. .
  • 3 bins (1 per genotype) are shown. 1 st bin: WBS; 2 nd bin: CTL; 3 rf bin: 7dupASD.
  • NCSC a and MSC
  • phase contrast microscopy shows a similar morphology between the four genotypes
  • DAPI stains nuclei
  • Flow cytometry analysis indicates a high percentage of HNKl -NGFR and CD73-CD44 double positive cells respectively in NCSC (d) and MSC lines (e).
  • FIG. 19 The WikiWilliams/7qllGB web platform. Representative screenshot of the WikiWilliams/7qllGB database as it appears to users searching for a specific gene of interest. All transcriptomic and genomic data presented in this paper as well as previously published datasets can be easily interrogated in a multi-layered format integrated with several biological databases.
  • fibroblast cell lines WBS1-2-3-4, 7Dup-ASD2, AtWBSl, CTL1R were obtained from Genetic Disease Biobank.
  • 7Dup-ASDl primary fibroblast was obtained from Azienda Ospedaliera-Universitaria "S. Maria della Misericordia", Perugia, Italy.
  • Fibroblasts were cultured in HF medium composed as follows: RPMI 1640, 1% L-Glutamine, 1% Pen-Strep, 15% FBS for few passages before reprogramming.
  • WBS1-2-3-4, 7Dup-ASDl-2, AtWBSl, CTL1R, and CTL2 fibroblast lines were reprogrammed using mRNA Reprogramming Kit (Stemgent).
  • mRNA Reprogramming Kit Stemgent
  • 7Dup-ASD2 and CTLIR lines microRNA Booster Kit (Stemgent) were used to enhance the reprograrnming.
  • CTL3 line was reprogrammed using STEMCCA polycistronic lentiviral vector followed by successful cre- mediated excision of the integrated polycistron.
  • epithelial-to-mesenchymal transition was monitored from day 5 by tracing GFP-positive cells.
  • Successfully reprogrammed colonies were assayed for pluripotency at day 20 using a live TRA- 1-60 antibody (Stemgent) and selected for further expansion as detailed below.
  • iPSC lines were cultured on mitomycin C-inactivated mouse embrionic fibroblasts (MEFs) as previously described 23 in a medium composed as follows: DMEM-F12 (Gibco) in a 1:1 ratio supplemented with 20% KSR, 1% Non Essential Amino Acids, 1% Pen-Strep, 1% Glutamine, 0.1 % beta-mercaptoethano 1, 10 ng/ml basic fibroblast growth factor (bFGF, Gibco). Colonies were passed and expanded twice by physical fractionation with a sterile needle and replated onto newly seeded MEFs for line establishment.
  • MEFs mitomycin C-inactivated mouse embrionic fibroblasts
  • iPSCs were adapted to grow in feeder-free condition on plates coated with human-qualified Matrigel (BD Biosciences) diluted 1:40 in DMEM-F12 and in mTeSR-1 (StemCell Technologies) medium and were passed by physical fractionation upon a 2 minutes treatment with Dispase (Sigma) at 37°C.
  • Feeder-free iPSCs were also adapted to grow in single cell culture by dissociating them by a 3 minutes treatment with Accutase (Sigma) at 37°C and finally resuspended in a suitable volume of mTeSR-1 supplemented with 5 uM Y-27632 (Sigma).
  • Teratoma assay was performed by subcutaneously injecting l-3xl0 6 iPSCs in human-qualified Matrigel (BD Biosciences) into the dorsal flanks of NOD-SCID IL2RG male mice. Teratomas were isolated when the diameter reached >1.5 cm and fixed in 4% buffered formalin. Samples were then OCT embedded, sectioned and stained for H&E and germ layer specific antibodies: desmin (Dako), S-100 (Dako) and cytokeratin (Dako).
  • Cells were fixed in 4% PFA for 20' and subsequently blocked in 10% FBS + 0,1% Triton for 30' at room temperature. Cells were incubated with primary antibodies overnight at 4°C and then with secondary antibodies for one hour at room temperature. Primary and secondary antibodies were resuspended in 10% FBS.
  • Genomic DNA was extracted from fibroblasts and feeder-free iPSC lines using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer specifications.
  • RNA was extracted from iPSC lines using the RNeasy Micro Plus Kit (Qiagen) according to manufacturer specifications, substituting the genomic DNA elimination column by needle and Dnase treatment (Qiagen). Quality and concentration of DNA and RNA was assessed using a NanoDiopSpectrophotometer (NanoDrop Technologies).
  • Proteins were extracted as follows: cells were scraped from the plate and centrifuged at 1100 g at 4°C for 3 minutes, then washed in PBS and lysed in RIP A buffer plus protease inhibitors cocktail (Sigma) on a spinning wheel at 4°C for 30 minutes. Lysates were sonicated using the Bioruptor Sonication System (UCD200) for 3 cycles of 30 seconds with 60 seconds breaks at high power. Lysates were centrifuged at 13000g for 15 minutes and supernatants were transferred to a new tube. Protein quantification was performed using the Bradford protein assay (BioRad) and following manufacturer instructions. Immunoblotting
  • Nanostring quantification was performed according to manufacturer instructions and data normalization was performed with the nSolver Analysis Software 1.1, using GAPDH, TUBB, and POLR1B as normalizers.
  • RNA sequencing was performed using Poly-A, RiboZero and Single Stranded kits (Illurnina) according to manufacturer instructions.
  • the inventors complemented their polyA dataset with the transcriptomic profiles of three control iPSC lines available from the literature (GEO samples GSM1153507, GSM1153512, and GSM1153513, fiorn cell lines ADRC40, WT- 9 and WT-126 respectively), which were selected on the basis of the similarity of their culture conditions and sequencing protocols to ours.
  • Reads were aligned to the hgl9 transcriptome using TopHat 2.0.10. The alignment was first performed on the RefSeq transcriptome and all the reads that had an edit distance > 1 were realigned on the genome, allowing a maximum read edit distance of 3 and 3 (100 bp reads) and 2 (50 bp reads) maximum mismatches. 50bp stranded reads (MSC transcriptomes, as well as transcriptomes of the knockdown and inhibited lines) were analyzed using the "fr-firststrand" option. Quantification of reads over the RefSeq transcriptome was performed with Cufflinks 2.2.1 using sequence-bias and multi-read corrections.
  • the inventors performed a series of differential expression analysis between randomly-selected samples, disarding comparisons in which the two groups were not balanced for sex and/or genotype. A minimum of 3 such combinations were tested per tissue, and the resulting genes were pooled for the purpose of enrichment analysis.
  • the inventors At the iPSC stage (using the polyA dataset), the inventors first randomly assigned all patients to two groups, but could the inventors obtain statistically significant genes in none of the combinations. The inventors therefore gradually removed patients until significant genes were obtained, which did not happen until a comparison involving 6 vs 6 samples (in each group, 3 samples from 2 patients). In contrast, when clones were selected and assigned to groups in a way that maximized the number of patients represented in each group, the inventors had to go down to 3 vs 3 (3 samples per group, coming from 3 different patients) to get statistically significant genes (18 DEGs, showing no significant GO enrichment). Similarly, random allocation of the control samples (including external controls, balanced across groups) yielded very few DEGs (maximum 17) and no significant GO enrichment.
  • NSC no significant differential expression
  • NPC very few genes
  • Retrotranscribed cDNAs have been obtained from 1 ⁇ % of total DNA-depleted RNA using the superscript VILO retrotranscription kit from Life Technologies according to manufacturer instructions.
  • the pair oligos used for qPCR were: GAPDH (F: GCACCGTCAAGGCTGAGAAC (SEQ ID No. 1), R: AGGGATCTCGCTCCTGGAA (SEQ ID No. 2)), BEND4 (F: GGAAAAGGAAAAGGTCAGTGC (SEQ ID No. 3), R: GTTTATCTGCTCTTCCGAGGG (SEQ ID No. 4)) and GTF2I (F: GATCTTGCAACCCTGAAATGG (SEQ ID No. 5), R: CACCTGGAGATAGTATTGACCTG (SEQ ID No. 6)).
  • iPSCs 10 8 iPSCs were used for each immunoprecipitation.
  • Cells were detached using accutase, resuspended in PBS containing 1% formaldehyde for fixation and quenched with 125 mM glycine.
  • Cells were lysed using ChIP lysis buffer (1% SDS, lOmM EDTA, 50mM Tris-HCl, pH 8.1) and sonicated using the Covaris Sonicator to generate 250 bp DNA fragments.
  • Soluble chromatin was diluted 10 times in ChIP dilution buffer (0.01% SDS, 1.1% Triton-XlOO, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl). Chromatin was incubated overnight at 4°C with the antibody and recovered the day after using Dynabeads Protein G (Life Technologies).
  • Beads were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, lmM EDTA, lOmM Tris-HCl, pH 8.1) and TE.
  • Low Salt Wash Buffer 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl
  • High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500m
  • Immunocomplexes were eluted in ChIP elution buffer (0.1% SDS, 1% Triton-XlOO, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl) and the decrosslinking was performed overnight at 65°C. DNA was purified using Qiaquick PCR Columns (Qiagen) according to manufacturer instructions.
  • DNA libraries were prepared according to Blecher-Gonenet al. 24 and DNA was sequenced on an Illumina HiSeq 2000 platform.
  • R CCACCGACCCTTTAGCTTTAG (SEQ ID No. 30)
  • TMEM20 F: GCTCCTGTAATTAGTGTCGGG (SEQ ID No. 31)
  • R GGGATCACTTTTCAGGGTCAG (SEQ ID No. 32)
  • peaks on the pooled reads from all samples (against the pooled inputs) to identify a broader set of putative binding sites, considering only regions with a fold enrichment over input of at least 5 and FDR ⁇ 0.001 (10691 peaks; 1554 target genes).
  • target genes were identified as genes with a peak in a -2.5kb/+lkb around their TSS.
  • Bivalent domains were defined as regions having H3K4me3 and H3K27me3 peaks within lkb of each other in ENCODE's data from the H9 human embryonic stem cell (hESC) line.
  • the inventors compared it to ENCODE's ChlPseq for H3K4me2 in HI hESC line. As expected, 95% of the LSD1 peaks defined by the inventors overlap with a H3K4me2 peak.
  • associations between TSS and distal enhancers were inferred from strong correlation, across ENCODE datasets, between the enhancer chromatin signature and expression at the putative target TSS; more specifically, the inventors considered only associations with a FDR ⁇ 0.001 and a minimum Pearson correlation of 0.5 with H3K27ac, H3K4mel, and -0.5 with DNA methylation, for a total of 5996 enhancer-TSS associations.
  • the beads are treated with one volume of 4X Novex LDS sample buffer (50 mM DTT) (Life Technologies) and the same volume of sample is loaded on a precast 12%-4% Novex gel (Life Technologies) for SDS-PAGE. Gels were stained using Colloidal Blue Coomassie (Fermentas). 7 discrete bands were cut and analyzed via liquid chromatography-tandem MS. Liquid chromatography-tandem MS (LC-MS/MS) analysis
  • Peptides separation was achieved on a linear gradient from 100% solvent A (5 % ACN, 0.1% formic acid) to 20% solvent B (acetonitrile, 0.1% formic acid) over 30 min and from 20% to 80% solvent B in 20 min at a constant flow rate of 0.3 uL/min on Agilent chromatographic separation system 1100 (Agilent Technologies, Waldbronn, Germany) where the LC system was connected to a 15 cm fused-silica emitter of 75 um inner diameter (New Objective, Inc. Woburn, MA USA), packed in-house with ReproSil-Pur C18-AQ 3 um beads (Dr. MaischGmbh, Ammerbuch, Germany) using a high-pressure bomb loader (Proxeon, Odense, Denmark).
  • Agilent chromatographic separation system 1100 Agilent chromatographic separation system 1100 (Agilent Technologies, Waldbronn, Germany) where the LC system was connected to a 15 cm fused-silica emitter of 75 um inner diameter (New Objective, Inc. Woburn
  • GTF2I knock-down were performed using validated pLKO.l TRC vector (TRCN0000019315 referred as sh2, TRCN0000364552 referred as sh52 and TRCN0000369208 referred as sh08).
  • a previously described pLKO.l TRC containing a scrambled short hairpin was used as a negative control 29 .
  • Viral particles were produced using psPAX2 and pMD2.G packaging vectors in 293T cells. Viral particles were harvested at 24 and 36 hours post-transfection and concentrated 250X by ultracentrifugation at 24000 g for 2 hours at 16°C. iPSC lines were infected with different amounts of viral particles, and the amount that gave rise to 50% of survival upon 1 ⁇ puromycin treatment for 72 hours was selected for following experiments.
  • Differentiation into the dorsal telencephalic lineage was accomplished by dual Smad inhibition in the presence of SB431542 (Tocris) and Noggin (R&D) 30 ' 31 .
  • Differentiation of NCSC and MSC was performed as previously described 32 , through the activation of Wnt signalling and Smad pathway blockade by administering the small molecules GSK3i (Calbiochem) and SB431542 (Tocris).
  • EBs Embryoid bodies production and LSD1 inhibitors treatments.
  • RNA extraction and cDNA preparation has been performed as described above.
  • LSD1 inhibitor has been also tested in non-differentiating condition according to the following experimental procedure: about 2,5x10 s cells were seeded at day 0 in feeder-fiee condition on a 6 cm (diameter) plate coated with diluted human-qualified Matrigel (BD Biosciences) and in mTeSR-1 (StemCell Technologies) medium supplemented with 5 uM Y-27632 (Sigma). mTeSR-1 was replaced at day 1 and day 2.
  • lxlO 6 cells were fixed in 4% PFA and subsequently blocked in 10% BSA. Cells were incubated for one hour with primary conjugated antibody resuspended in 1-2% BSA.
  • the primary conjugated antibody used were CD57-FITC (HNKl, BD), CD271-647 (NGFR, BD), CD44- APC (EBIOS) and CD73-PE (BD). Analysis were performed on FACSCalibur (BD Biosciences) and data were analyzed with FCS express software (Tree Star inc.).
  • DNA was isolated from parental fibroblast and iPSC using Qiagen kit as described above. DNA concentration and purity were determinate with a ND-1000 spectrophotometer (NanoDrop Technologies, Berlin, Germany) while whole-genome copy number variations (CNVs) analysis was carried out using the CytoScan HD array platform (Affymetrix, Santa Clara, CA). The CytoScan HD assay was performed according to the manufacturer protocol, starting with 250 ng of DNA. Briefly, total genomic DNA was digested with a restriction enzyme (Nspl), ligated to an appropriate adapter for the enzyme and subjected to PCR amplification using a single primer.
  • Nspl restriction enzyme
  • the PCR products were labeled with a biotinylated nucleotide analogue, using terminal deoxynucleotidyl transferase (TdT) and hybridized to the microarray.
  • TdT terminal deoxynucleotidyl transferase
  • Microarrayanalysis was performed with the Affy package using Marc Carlson's Hugene 2.1st RefSeq annotation file, version 18. Background normalization was performed using the RMA method, whereas between-sample normalization was performed using the quantile normalization method. Quantification of expression was obtained using perfectly matching probes only with medianpolish summarization, averaging probesets to obtain gene-level expression. Probesets not assigned to known genes or having log2 fold changes ⁇ 0.5 were discarded and differential expression was assessed using a 2-tailed t-test. For the purpose of enrichment analyses, DEGs with a FDR ⁇ 0.2 were considered.
  • RNAseq data enrichment analysis was performed using the R package GOseq in order to correct for transcript length bias considering only categories with at least 10 annotated genes and discarding categories that had less than 8 significant genes.
  • the enrichment analysis was performed with the package TopGO using the classic algorithm and Fisher's test with the same cutoffs described above.
  • parent categories that had enriched children were first removed and then maps were created with the package Treemap, using as colors the combination of non-overlapping parent categories accounting for the largest proportion of plotted categories. All reported FDR values were calculated using the Benjamini-Hochberg method.
  • Colonies occupying approximately half of the surface of the 96 well are split by washing cells with 100 ul PBS and then incubating them with 30 ul of Gentle dissociation reagent for 3 minutes at 37°C. Gentle dissociation reagent is discarded and cells are roughly resuspended in 200 ul mTeSR. Resuspension is very brief so as to avoid breaking colony clumps into single cells. Resuspended clumps are then plated in 48-well plate wells, in a total volume of 300 ul of mTeSR without Rock Inhibitor per well,
  • mice laminin (0.2 mg/1)
  • Doxycycline (2 mg/1) is added preferably on the same day to induce NGN2 and retained in the medium until the end of the experiment,
  • hESC-qualified matrigel diluted 1:40 in DMEM/F12/Gln/Pen-Strep
  • Cells are always incubated at 37°C with 3% 02 and 5% C02.
  • Cells are always split using accutase and plated in mTeSR supplemented with 5 uM Rock Inhibitor, which is then removed after the first media change.
  • the method optionally comprises extraction of genomic DNA from each stable cell line to quantify by digital PCR or TaqMan the exact amount of UbC-rtTA and TetO-NGN2-EGFP- Puro transgene copy numbers.
  • the present method allows to:
  • EXAMPLE 1 Establishment of a large cohort of transgene-free induced pluripotent stem cell lines from WBS and 7dupASD patients
  • the inventors selected a highly informative cohort of WBS and 7dupASD patients, whose fibroblast biopsies were deposited in the Genomic and Genetic Disease Biobank (ht ⁇ ://www.telethon.it/en/scientists ⁇ iobanks) and who were assessed by a multidisciplinary team of specialists for a detailed clinical record (Fig. la and Table 3).
  • This cohort includes: i) four patients carrying the typical WBS deletion; ii) one patient carrying an atypical WBS deletion that spares several genes including BAZ1B, who exhibits milder craniofacial dysmorphisms 35 and lack cardiovascular abnormalities, supporting a role for BAZ1B in neural crest-derived lineages 36 ; iii) two patients carrying the typical duplication of the 7qll.23 interval associated to language impairment, autism spectrum disorder and craniofacial dysmorphisms 37 ; and iv) one unaffected relative of a typical WBS patient, chosen as genetically half-matched control (three iPSC lines from two additional unrelated individuals were included as additional controls, Fig. la).
  • the inventors characterized 3 independent iPSC lines from each patient or unaffected relative, along with 2 independent iPSC lines from the unrelated control individual, and one additional iPSC line previously reprogrammed by a conditional lentiviral vector following Cre-mediated excision of the single copy integrant amounting to a total cohort of 27 independent iPSC lines (Fig. la).
  • iPSC lines exhibited typical morphology, expressed the full range of pluripotency markers including ALP (alkaline phosphatase), OCT4, NANOG, Tra-1-60, SOX2 and SSEA-4, (Fig. 12c-d) and contributed to the three embryonic germ layers upon teratoma differentiation in vivo (Fig. 12c).
  • iPSC lines were also assessed for genomic integrity by comparison to the respective parental fibroblasts through high density CytoScan Arrays. As shown in Table 4, virtually all iPSC lines carried copy number variants. Several of these were acquired de novo during the reprograrnming process, as an inherent aspect of the attending laboratory manipulations (such as the use of reprograrnming factors and the trigger of sustained proliferation), and as such distinguish them univocally from the parental somatic cells originally sourced from the research participants. Others instead pre-existed already in parental fibroblasts, consistent with recent evidence of pronounced CNV mosaicism in human skin from which 'de novo ' iPSC-specific CNV emerge and become detectable as a result of clonal expansion 40 .
  • the iPSC from this cohort are clearly distinct both from: i) any other iPSC reported thus far, due to the unique genetic make up that includes reprogramming-induced specific CNVs, and to the method employed for reprograrnming; ii) any somatic cell originally sourced from the research participants, due to the specific CNVs that were introduced during reprograrnming.
  • EXAMPLE 2 Expression of 7qll.23 genes follows gene dosage in the pluripotent state
  • the inventors In order to ascertain whether the pluripotent state represented a meaningful stage at which to probe the effect of 7ql 1.23 dosage, the inventors first asked whether the mRNA expression of the 7ql 1.23 genes follows gene dosage. For this the inventors resorted to the high accuracy of Nanostring-based quantitation as well as to RNAseq and found that the expression of all genes of the interval (including those expressed at very low levels) mirrors gene dosage (Fig. lc and Fig. 13a), thus excluding compensatory effects from the wild type allele.
  • EXAMPLE 3 7qll.23 dosage imbalance causes transcriptional dysregulation in disease- relevant pathways already at the pluripotent state
  • Figure 2b shows a treemap representation of the most specific enriched biological processes, in which square sizes are proportional to the significance of the enrichment (GO enrichments for each comparison are shown in Fig. 14a-b-c).
  • the top-ranking categories are related to extracellular matrix (EM) organization (FDR -2.3E-15), which appears especially significant in light of the wide range of connective tissue alterations that characterize WBS, and to the nervous system (e.g. neuron differentiation FDR ⁇ 2.7E-7, neuron projection development FDR -3.6E-6), providing a molecular context for the defining neurodevelopmental features of the two conditions.
  • EM extracellular matrix
  • FDR -2.3E-15 extracellular matrix organization
  • FIG. 14b-c shows a treemap representation of the most specific enriched biological processes, in which square sizes are proportional to the significance of the enrichment (GO enrichments for each comparison are shown in Fig. 14a-b-c).
  • the top-ranking categories are related to extracellular matrix (EM) organization (FDR -
  • cellular calcium ion homeostasis FDR -0.0012
  • FDR -0.0012 cellular calcium ion homeostasis
  • DEGs in category: ATP1A2, ATP2A2, ATP7B, CACNA1A, CALCR, CAV1, CCDC47, CCKBR, CD40, DIAPHl, EDNRB, EPHX2, GRIK2, GSTOl, GTF2I, HRC, ITPR3, KDR, RYR1, RYR2, SLC30A1, SLC8A3, TACR1, TRDN DEGs in category: ATP1A2, ATP2A2, ATP7B, CACNA1A, CALCR, CAV1, CCDC47, CCKBR, CD40, DIAPHl, EDNRB, EPHX2, GRIK2, GSTOl, GTF2I, HRC, ITPR3, KDR, RYR1, RYR2, SLC30A1, SLC8A3, TACR1, TRDN);
  • inner ear morphogenesis (FDR -0.0046), consistent with the combination of hyperacusis and sensorineural hearing loss that is virtually always present in WBS 47 , as well as with the balance and sensory processing disorders found in ASD 48 (DEGs in category: COL11A1, COL2A1, FZD2, GBX2, MAFB, MY06, NTN1, SOX9, WNT3A, ZEB1, ZIC1);
  • iii) a number of categories relevant for the craniofacial phenotype, such as skeletal muscle organ development (FDR ⁇ 0.0067), migration (FDR ⁇ 4.2E-8) and neural crest cell differentiation (FDR ⁇ 0.011) (DEGs in any of the three categories: ;
  • iv) categories such as blood vessel development (FDR ⁇ 0.0068) and cardiovascular system development (FDR ⁇ 0.0043), that reflect the wide range of cardiovascular problems in WBS; v) kidney epithelium development (FDR ⁇ 0.0026), in line with the highly prevalent kidney abnormalities of WBS 49 .
  • the set of symmetrically dysregulated genes includes genes associated to characterizing phenotypes of the 2 conditions, as in the case of PDLIM1 and MYH14, the former associated to attention-deficit disorder 50 , neurite outgrowth 51 , cardiovascular defects, and hyperacusis, and the latter involved in hearing impairment 31 .
  • EXAMPLE 4 GTF2I dosage accounts for a significant fraction of transcriptional dysregulation in the pluripotent state.
  • GTF2I assembles a transcriptional co-repressor complex containing LSD1 and HDAC2
  • the inventors purified its protein complex and defined its genome-wide occupancy.
  • the inventors tested the ability of two GTF2I antibodies to immunoprecipitate GTF2I in its native form (Fig. 16a) and selected the most efficient ones for large-scale IP (immunoprecipitation) on one representative iPSC line for each genotype, followed by Mass Spectrometry (MS) of discrete bands from IP eluates and validation of selected candidates (Fig. 3a-b).
  • MS Mass Spectrometry
  • the inventors found two transcriptionally repressive chromatin modifiers, the histone demethylase LSD1 (also known as KDM1A) and the histone deacethylase HDAC2 (Q92769), that were sub-stoichiometrically associated to GTF2I.
  • the inventors identified two transcription factors, ZMYM2 and ZMYM3 (A6NHB5), previously reported as part of LSD 1 -containing transcriptional complexes (BHC complex) 55"57 .
  • the inventors also found the co-repressor RCOR2 (Q8IZ40), recently reported, in place of RCOR1, as the main partner of LSD 1 in the pluripotent state 58 .
  • the other proteins identified in the complex are DNMT3B (PI 1388-4) and FXR1 (P51114).
  • EXAMPLE 6 Identification of GTF2I targets in WBS and 7DupASD patient-derived iPSCs
  • Fig. 16c the antibody with the highest signal-to-noise ratio
  • TSS transcription start site
  • the 436 genes are as follows:
  • GTF2I core targets (conserved promoter peak in all control samples):
  • GTF2I targets differentially-expressed in iPSC:
  • WBS lines retain only a subset of these core GTF2I targets, consistent with a progressive dosage-dependent loss of GTF2I occupancy.
  • the inventors next confirmed by ChlP-qPCR (ChIP coupled with quantitative real-time PCR) dosage-dependent occupancy on a subset of core GTF2I targets. Additionally, they validated a generally lower enrichment, in WBS, for dosage sensitive targets, with levels of binding comparable to negative control regions (Fig. 16d).
  • GTF2I binds preferentially to bivalent domains (p- value ⁇ l,58e-03 for core targets and 1.13e-10 for all targets).
  • the distribution of GTF2I binding vis-d-vis the TSS does not correlate with expression (Fig. 3e, inner pie).
  • the inventors uncovered in iPSC are GTF2I-bound (-9%, with 3% being core GTF2I targets), and in turn only -5% of the GTF2I targets are differentially expressed.
  • DEGs within the proportion of DEGs that the inventors found attributable to GTF2I upon its knockdown, only 8% appear to be direct targets.
  • GTF2I dosage-dependent transcriptional dysregulation could be mediated by distal binding, looking at GTF2I-bound enhancers associated to putative target genes.
  • the inventors found however only one of the 89 GTF2I- bound enhancers associated to a GTF2I-attributable, differentially-expressed TSS (PHC2), thus excluding that GTF2I acts mostly at enhancers, and supporting the alternative model of a largely indirect impact mediated by a minority of GTF2I direct targets.
  • BEND4 a TF that is both highly expressed in the brain and is part of a large CNV that has been reported in an ASD patient 62 .
  • BEND4 was confirmed to be differentially expressed both between genotypes (Fig. 4a-b) and upon GTF2I knockdown using several independent short-hairpin expressing vectors (Fig. 4c-d-e).
  • BEND4 and GTF2I levels are significantly anticorrelated in the human brain (Pearson coefficient of -0.71 between regional averages) according to a very recent compendium of gene expression across several brain regions from over a hundred individuals 63 (a weaker but significant correlation was also found among the regions of the Allen Brain Atlas) 64 .
  • the inventors decided to profile LSD1 occupancy in iPSCs in order to test whether it could discriminate dosage-dependent and independent GTF2I targets.
  • Comparison between the GTF2I and LSD1 profiles revealed that while GTF2I co-occupies only a relatively small fraction of the LSD1 targets (Fig. 3f), the inventors were able to detect significant LSD1 peaks on 53% of the GTF2I conserved peaks (Fig. 3g).
  • LSD 1 -bound GTF2I dosage-sensitive targets include further disease-relevant genes, as in the paradigmatic case of CRB2 (Fig. 16d), which is implicated in cleft palate and lip development 65 , and whose dosage sensitivity could provide a molecular link to the symmetrical phyltrum defects (respectively expanded or reduced) that are observed in WBS and 7dupASD patients.
  • LSD 1 -sensitive targets also include critical genes that have already been causally associated to ASD, such as NLGN2, MAP2, and PRICKLEl.
  • RHBDF2 has a GTF2I peak on its promoter.
  • EXAMPLE 7 iPSC-specific transcriptional deregulation is amplified during development in a lineage-specific manner
  • iPSC lines into three lineages of cardinal relevance for the two conditions (Fig. 5a): i) Pax6 + telencephalic neural stem cells and progenitors (NPC) 30 ' 31 ; ii) neural crest stem cells (NCSC) 32 , which originate the craniofacial structures along with several other disease-relevant lineages; iii) mesenchymal stem cells (MSC) 32 , hierarchically upstream of osteocytes, chondrocytes, smooth muscle cells, and other physiopathologically meaningful cell types.
  • NPC neural crest stem cells
  • MSC mesenchymal stem cells
  • the inventors confirmed that patient-derived NPCs expressed key fbrebrain markers such as FOXG1, OTX2 and ZOl, and were typically arranged in neural rosettes with TBR2 + intermediate progenitors surrounding apical Pax6 + progenitors (Fig 5b).
  • the inventors profiled 15 patient and control-derived NPC lines and found that most of the differentially expressed genes were enriched in GO categories related to neuronal function such as axon guidance, regulation of transmitter secretion and negative regulation of axonogenesis (Fig. 5c).
  • the inventors differentiated the same cohort of iPSC lines into NCSC that displayed distinct morphology (Fig.
  • iPSC-primed transcriptional dysregulation upon further differentiation, the inventors induced patient-derived NCSC towards the MSC fate, characterized by a typical MSC-like morphology (Fig. 17b) and the expression of key MSC markers CD44 and CD73 ( ⁇ 95%) (Fig. 5f and Fig. 17e).
  • Transcriptomic profiling confirmed that, with the notable exception of ELN, also in MSC expression of 7ql 1.23 genes recapitulated dosage (Fig. 17f), and yielded 422 DEGs showing enrichment for several GO categories related to tissue morphology (Fig. 5g).
  • IPA Ingenuity Pathway Analysis
  • iPSC DEGs conserved for the GO categories that had been found enriched already in iPSC.
  • iPSC DEGs are preferentially retained by category in a lineage-appropriate manner so that, for each target lineage, the proportion of conserved iPSC DEGs is much greater in categories relevant to that lineage (such as axonogenesis and axon guidance in the neural lineage, synapse-related catetgories in NCSC that will originate the peripheral nervous system and smooth muscle related categories in MSC).
  • EXAMPLE 8 A web platform for 7qll.23 CNV syndromes
  • WikiWilliams-7qGeneBase a new web platform called WikiWilliams-7qGeneBase to make their data accessible to the community of scientists and practitioners working on these two diseases, through a user-friendly, gene-centered interface.
  • WikiWilliams is open to contributions by other groups through submission to the database's curators, with the aim of assembling in one site all molecular data on 7qll.23 syndromes (Fig. 7b and Fig. 19).
  • the platform is openly accessible at http:/foio.ieo.eu/wbs/.
  • EXAMPLE 9 An approach to isolate iPSC-derived FOXGl-expressing cortical progenitors
  • Fig. 8 The proof of principle of experiments aimed at isolating cortical progenitors by selection and FACS sorting is described in Fig. 8. Briefly, the inventors designed a lentiviral construct that expresses GFP from a ubiquitous promoter and the puromycinr esistance gene under the control of the FOXG1 promoter. The figure shows the infection of a control iPSC line (reprogrammed from a WBS patient relative) with different concentrations of this lentiviral vector, and the assessment of infection efficiency by GFP fluorescence (both by immunofluorescence and FACS).
  • EXAMPLE 10 The screening assay based on qRT-PCR
  • the inventors have already validated the automated workflow for the scoring of the expression of GTF2I and BAZ1B (by multiplex qRT-PCR) in iPSC grown on a 96-well format (Fig. 9). This is the format in which the screening will be conducted.
  • the inventors were able to measure consistently the levels of GTF2I across 3 different concentrations of seeded iPSC (from 6.000 to 12.000), finding the expected linear relationship between number of cells and GTF2I expression, with excellent consistentcy across different wells of the same plate. Even across wells from different plates, qRT-PCR measurements are extremely consistent.
  • the quantification shows a minimal coefficient of variation (CV) across both intra- and inter- plate measurements.
  • the inventors modified the protocol for the direct differentiation of iPSCs towards the cortical glutamatergic lineage via induction of a lentivirally transduced factor, NGN2 34 .
  • the original protocol employs two lentiviral vectors to integrate i) the inducible tetracyclin transactivator (rtTA) constitutively expressed under the UbC promoter and ii) an NGN2-EGFP- Puro polypeptide-econding cDNA whose transcription is driven by the transactivator in the presence of doxycylin.
  • rtTA inducible tetracyclin transactivator
  • NGN2-EGFP- Puro polypeptide-econding cDNA whose transcription is driven by the transactivator in the presence of doxycylin.
  • Clones are established by: i) sorting a co-infected iPSC population into a 96-well plate; ii) selecting and expanding colonies with an ES-like (undifferentiated) morphology, iii) splitting selected colonies in two wells and testing one for GFP expression upon induction; iv) expanding and stocking 3-5 GFP positive iPSCs lines.
  • the inventors chose the paradigmatic pair of genetic syndromes caused by symmetrical 7qll.23 CNV 7qll.23 in order to test the potential of iPSCs for defining how gene dosage impacts disease-relevant pathways in early developmental lineages. Their findings have broad significance for the molecular pathogenesis of WBS and 7dupASD as well as for the reprogramming-based disease modeling field as a whole.
  • the size and quality of the present invention's iPSC cohort permitted two key observations on the molecular impact of 7qll.23 CNV: i) that these caused significant transcriptional dysregulation in disease-relevant pathways already in the pluripotent state; and ii) that this dysregulation was selectively amplified in a lineage-specific manner, with disease- relevant pathways preferentially and progressively more affected in differentiated lineages matching specific disease domains.
  • the significance of this observation for the iPSC modeling field lies in the fact that the pluripotent state is by far the best characterized and most standardized one among the human developmental stages captured in vitro. Importantly, it is also the most amenable to high-throughput upscaling.
  • the pluripotent state is not only a viable stage in which to measure disease-relevant transcriptional effects of genetic alterations, but that these effects are also predictive of further dysregulation in differentiated lineages, grounds the feasibility of middle-to-high-throughput iPSCs characterization in order to functionally annotate human genomes, prior to selecting lines and assays for more labor-intensive differentiation courses.
  • GTF2I assembles a chromatin repressive complex with LSD1 and HDAC2 and binds preferentially to bivalent chromatin promoters, whose timed resolution has been shown by us and several other groups to be essential for normal development, especially along the neural lineage 77"80 .
  • LSD1 inhibitors and/or HDAC2 inhibitors may be used for the prevention and/or treatment of a neurodevelopmental disorder entailing intellectual disability (ID) and/or belonging to the Autism Spectrum Disorder (ASD) and/or Schizophrenia (SZ).
  • the inventors provide a user-friendly, open source web platform in which the inventors assembled the multi-layered datasets from this first cohort of WBS and 7dupASD samples, and which was designed to integrate ongoing contributions from the entire scientific community working on these two diseases, thus serving also as a first template for data sharing from iPSC- based functional genome annotation.
  • Van der Aa, N. et al Fourteen new cases contribute to the characterization of the 7qll.23 microduplication syndrome. Eur J Med Genet 52, 94-100 (2009).
  • Campuzano, V. et al Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome.
  • Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4).
  • DFNA4 autosomal dominant hearing impairment
  • Hedgehog acyltransferase Hhat
  • Hedgehog signaling resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects.
  • Dobreva, G. et al SATB2 is a multifunctional determinant of craniofacial patterning and osteoblast differentiation. Cell 125, 971-86 (2006).

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Abstract

La présente invention concerne une cellule souche pluripotente induite (CSPi) produite à partir d'un fibroblaste obtenu auprès d'un sujet atteint d'un trouble neurologique du développement entraînant une déficience intellectuelle (ID) et/ou d'un trouble appartenant au trouble du spectre autistique (ASD) et/ou de la schizophrénie (SZ) et leurs utilisations. La présente invention concerne également une cellule progénitrice neuronale corticale ou une cellule neuronale glutamatergique ou gabaergique corticale à terminaison différenciée s ou une lignée de cellules souches de crête neurale, une lignée de cellules souches mésenchymateuses produite à partir de la CSPi ou la lignée de CSPi. L'invention concerne également un procédé permettant d'identifier un composé pour le traitement et/ou la prévention d'un trouble neurologique du développement entraînant une déficience intellectuelle (ID) et/ou d'un trouble appartenant au trouble du spectre autistique (ASD) et/ou de la schizophrénie (SZ) et un inhibiteur de LSD1 ou un inhibiteur de HDAC 2 utiles dans le traitement de tels troubles.
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US20210332436A1 (en) * 2018-11-13 2021-10-28 Memorial Sloan-Kettering Cancer Center Stem cell-based multiplex methods and compositions
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